Epstein Barr Virus (EBV) latent infection has been etiologically linked to human malignancies of lymphoid and epithelial cell lineage. In latently infected cells EBV DNA persists as a multicopy episome that maintains a stable copy number during cellular proliferation. The latent virus expresses a limited set of viral genes that contribute to host-cell growth transformation and viral genome survival. EBNA1 is a latency-associated gene product that is consistently expressed in all EBV-associated tumor cells and is essential for latent genome maintenance. EBNA1 stimulates DNA replication and plasmid maintenance when bound to the viral origin of plasmid replication OriP. The mechanism of EBNA1-dependent replication and plasmid maintenance is not completely understood. In this application, we focus on the functions of EBNA1 in viral DNA replication and plasmid maintenance. In previous studies, we found that EBNA1 binds cooperatively with telomere repeat factor 2 (TRF2) at the dyad symmetry (DS) element of OriP, and that TRF2 contributes to both DNA replication and plasmid maintenance function. New data suggest that TRF2 may contribute to the recruitment of the Origin Recognition Complex (ORC) and the intra-S phase checkpoint proteins important for OriP replication and maintenance. In this revised competitive renewal, we focus on the role of EBNA1 at OriP, and in particular the function of the RGG motifs in EBNA1 that have been genetically implicated in both replication and maintenance function (aim 1). We found that EBNA1 RGG motifs recruit ORC in an RNA-dependent manner. We also found that TRF2 recruits S phase checkpoint proteins to OriP, and explore their contribution to episome replication and maintenance. In particular, we explore the hypothesis that recombination junctions form at OriP, and that these junctions are important for sister-chromatid cohesion and plasmid segregation (aim 2). Finally, we investigate the mechanism of hydroxyurea-induced loss of EBV episomes, and how this may reveal critical mechanisms regulating OriP replication and maintenance (aim 3). These aims are integrated by a central hypothesis that episome maintenance and plasmid segregation are mechanistically coupled to DNA replication and the nucleoprotein structures that form as a byproduct of DNA replication through OriP. These aims are further integrated by the long-term goal of identifying critical viral and cellular targets for inhibiting DNA replication and genome maintenance of Epstein-Barr virus latent infections. PUBLIC HEALTH RELEVANCE: EBV latent infection has been implicated in multiple human cancers. At present, there is no effective treatment for the latent viral infection. Disruption of episomal maintenance or viral latent cycle replication is likely to prevent EBV-associated disease. We have focused on novel aspects of episomal maintenance that have emerged from our published work and preliminary studies. Here we test the hypothesis that EBV episome maintenance depends on the telomere repeat factors and the recruitment of S phase checkpoint proteins to OriP. The S phase checkpoint proteins provide a previously unappreciated mechanisms for regulation of EBV episomal maintenance during latency. We provide data that they are the target of hydroxyurea treatment, which is the only documented treatment for eliminating latent viral episomes. We propose that the further characterization of this mechanism will provide new targets for pharmacological intervention in EBV-associated disease.